Compositions for the detection of proteases in biological samples and methods of use thereof

ABSTRACT

The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone each end of which is conjugated to a fluorophore. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.

FIELD OF THE INVENTION

This invention pertains to a class of novel fluorogenic compositionswhose fluorescence level increases in the presence active proteases.These fluorogenic protease indicators typically fluoresce at visiblewavelengths and are thus highly useful for the detection andlocalization of protease activity in biological samples.

BACKGROUND OF THE INVENTION

Proteases represent a number of families of proteolytic enzymes thatcatalytically hydrolyze peptide bonds. Principal groups of proteasesinclude metalloproteases, serine proteases, cysteine proteases andaspartic proteases. Proteases, in particular serine proteases, areinvolved in a number of physiological processes such as bloodcoagulation, fertilization, inflammation, hormone production, the immuneresponse and fibrinolysis.

Numerous disease states are caused by and can be characterized byalterations in the activity of specific proteases and their inhibitors.For example emphysema, arthritis, thrombosis, cancer metastasis and someforms of hemophilia result from the lack of regulation of serineprotease activities (see, for example, Textbook of Biochemistry withClinical Correlations, John Wiley and Sons, Inc. N.Y. (1993)).

Similarly, proteases have been implicated in cancer metastasis.Increased synthesis of the protease urokinase has been correlated withan increased ability to metastasize in many cancers. Urokinase activatesplasmin from plasminogen which is ubiquitously located in theextracellular space and its activation can cause the degradation of theproteins in the extracellular matrix through which the metastasizingtumor cells invade. Plasmin can also activate the collagenases thuspromoting the degradation of the collagen in the basement membranesurrounding the capillaries and lymph system thereby allowing tumorcells to invade into the target tissues (Dano, et al. Adv. Cancer. Res.,44: 139 (1985).

Clearly measurement of changes in the activity of specific proteases isclinically significant in the treatment and management of the underlyingdisease states. Proteases, however, are not easy to assay. Typicalapproaches include ELISA using antibodies that bind the protease or RIAusing various labeled substrates. With their natural substrates assaysare difficult to perform and expensive. With currently availablesynthetic substrates the assays are expensive, insensitive andnonselective. In addition, many "indicator" substrates require highquantifies of protease which results, in part, in the self destructionof the protease.

Recent approaches to protease detection rely on a cleavage-inducedspectroscopic change in a departing chromogen or fluorogen located inthe P1' position (the amino acid position on the carboxyl side of thecleavable peptide bond) (see, for example U.S. Pat. Nos. 4,557,862 and4,648,893). However, many proteases require two or three amino acidresidues on either side of the scissile bond for recognition of theprotease and thus, these approaches lack protease specificity.

Recently however, fluorogenic indicator compositions have been developedin which a "donor" fluorophore is joined to an "acceptor" chromophore bya short bridge containing a (7 amino acid) peptide that is the bindingsite for an HIV protease and linkers joining the fluorophore andchromophore to the peptide (Wang et al. Tetra. Letts. 45: 6493-6496(1990)). The signal of the donor fluorophore was quenched by theacceptor chromophore through a process of resonance energy transfer.Cleavage of the peptide resulted in separation of the chromophore andfluorophore, removal of the quench and a subsequent signal from thefluorophore.

Unfortunately, the design of the bridge between the donor and theacceptor led to relatively inefficient quenching limiting thesensitivity of the assay. In addition, the chromophore absorbed light inthe ultraviolet range reducing the sensitivity for detection inbiological samples which typically contain molecules that absorbstrongly in the ultraviolet.

Clearly fluorogenic protease indicators that show a high signal levelwhen cleaved, and a very low signal level when intact, that show a highdegree of protease specificity, and that operate exclusively in thevisible range thereby rendering them suitable for use in biologicalsamples are desirable. The compositions of the present invention providethese and other benefits.

SUMMARY OF THE INVENTION

The present invention provides for novel reagents whose fluorescenceincreases in the presence of particular proteases. These fluorogenicprotease indicators provide a high intensity fluorescent signal at avisible wavelength when they are digested by a protease. Because oftheir high fluorescence signal in the visible wavelengths, theseprotease indicators are particularly well suited for detection ofprotease activity in biological samples, in particular, in frozen tissuesections.

The fluorogenic protease indicators of the present invention arecompositions suitable for detection of the activity of a protease. Thesecompositions have the general formula: ##STR1## in which P is a peptidecomprising a protease binding site consisting of about 2 to about 8,more preferably about 2 to about 6 and most preferably about 2 to about4 amino acids; F¹ and F² are fluorophores; S¹ and S² are peptide spacersranging in length from 1 to about 50 amino acids; n and k areindependently 0 or 1; and C¹ and C² are conformation determining regionscomprising peptides ranging in length from about 1 to about 3 aminoacids. The conformation determining regions each introduce a bend intothe composition thereby creating a generally U-shaped configuration ofthe composition in which the fluorophores are adjacent to each otherwith a separation of less than about 100 Å. When either of the spacers(S¹ and S²) are present they are linked to the protease binding site bya peptide bond to the alpha carbon of the terminal amino acid. Thus,when n is 1, S¹ is joined to C¹ by a peptide bond through a terminalalpha amino group of C¹, and when k is 1, S: is joined to C² by apeptide bond through a terminal alpha carboxyl group of C².

In a preferred embodiment, protease binding site (P) is a tetrapeptide,C¹ is a tripepride and C² is an amino acid or a dipeptide. Thecomposition thus has the formula: ##STR2## where C¹ ₅, C¹ ₄, C¹ ₃, P₂,P₁, P₁ ', P₂ ' are amino acids, and Y is a composition selected from thegroup consisting of compounds of formulas: ##STR3## where C² ₃, C² ₃ areamino acids.

In a particularly preferred embodiment, the composition is a compositionof formula II in which the amino acid composition of P corresponds tothe 4 amino acids symmetrically disposed adjacent to the cleavage site(the P₁ -P₁ ' peptide bond) cut by a particular protease. Theconformation determining regions (C¹ and C²) then comprise the remainingresidues of the protease binding site regions. Some of these C¹ and C²amino acids, however, may be substituted according to one or more of thefollowing guidelines:

1. If the P₂ ' site is not a proline then C² is a dipeptide (FormulaIII) Pro-Cys or Aib-Cys, while conversely, if the P₂ ' site is a prolinethen C² is a single amino acid residue (Formula IV) Cys.

2. If the P₂ site is not a proline then C¹ is a tripeptide consisting ofAsp-C¹ ₄ -Pro, Asp-C¹ ₄ -Aib, Asp-Aib-Pro, Asp-Pro-C¹ ₃, Asp-Pro-Aib, orAsp-Aib-Aib, while if the P₂ site is a proline residue then group C¹ isa tripeptide consisting of Asp-C¹ ₄ -C¹ ₃ or Asp-C¹ ₄ -Aib.

3. If the P₃ residue is a proline then C¹ is a tripeptide consisting ofAsp-C¹ ₄ -Pro or Asp-Aib-Pro.

4. If the P₄ residue is a proline then C¹ is a tripeptide consisting ofAsp-Pro-C¹ ₃ or Asp-Pro-Aib.

5. If P₂ and C¹ ₃ are both not prolines then C¹ is a tripeptideconsisting of Asp-Pro-C¹ ₃, Asp-Aib-C¹ ₃, Asp-C¹ ₄ -Pro, Asp-C¹ ₄ -Aib,Asp-Pro-Aib, or Asp-Aib-Pro.

Table 2 indicates particularly preferred protease binding sites (P) andsuitable conformation determining regions (C¹ and C²). Thus, thisinvention also includes compositions of formula II in which P₂ is Pro,Gly, Phe, Arg, Leu, Gln, Glu, Ile, or Ala; P₁ is Cys, Met, Nle, Arg,Leu, Gly, His, Glu, Ala, or Asn; P₁ ' is Thr, Ser, Met, Nle, Leu, Ala,Ile, Phe, Val, Glu, or Tyr; and P₂ ' is Ile, Gly, Met, Nle, Leu, Ala,Gln, Arg, Val or Asn. Particularly preferred are compositions of formulaII in which the P domain (P₂ -P₁ -P₁ '-P₂ ') is Pro-Met-Ser-Ile,Pro-Nle-Ser-Ile, Gly-Arg-Thr-Gly, Arg-Met-Ser-Leu, Arg-Nle-Ser-Leu,Gly-Arg-Ser-Leu, Leu-Leu-Ala-Leu, Leu-Gly-Ile-Ala, Gln-Gly-Ile-Leu,Gln-Gly-Leu-Leu, Gln-Gly-Ile-Ala, Gln-Ala-Ile-Ala, Glu-Gly-Leu-Arg,Gly-His-Phe-Arg, Leu-Glu-Val-Met, Leu-Glu-Val-Nle, Ile-His-Ile-Gln,Ala-Asn-Tyr-Asn, Ala-Gly-Glu-Arg, or Ala-Gly-Phe-Ala, Gln-Gly-Leu-Ala,Ala-Gln-Phe-Val, Ala-Gly-Val-Glu, Phe-Cys-Met-Leu, Phe-Cys-Nle-Leu(Sequence ID Nos. 1-25, respectively).

This invention also includes compositions of formula II in which C¹ ₅ isAsp; C¹ ₄ is Ala, Met, Nle, Aib, Pro, Ile, Gly, Asp or Arg; and C¹ ₃ isIle, Aib or Pro. Particularly preferred are compositions in which the C¹conformation determining region (C¹ ₅ -C¹ ₄ -C¹ ₃) is Asp-Ala-Ile,Asp-Ile-Pro, Asp-Aib-Pro, Asp-Gly-Pro, Asp-Asp-Pro, Asp-Arg-Pro,Asp-Ile-Aib, Asp-Aib-Aib, Asp-Gly-Aib, Asp-Asp-Aib or Asp-Arg-Aib.

Preferred protease indicators include compositions of formula II inwhich P₂ ' is a Pro, in which case C² is a single amino acid. Thus Y isformula IV in which C² ₃ is preferably a Cys. In another embodiment, Yis formula Ill and C² ₃ is Pro and C² ₄ is Cys.

In a particularly preferred embodiment F¹ is a fluorophore having anexcitation wavelength ranging from 315 nm to about 650 nm, F² is afluorophore having an excitation wavelength ranging from 315 nm to about650 nm or both F¹ and F² are fluorophores having excitation andabsorbtion wavelengths ranging from 315 nm to about 650 nm. F¹ may bethe donor fluorophore with F² the acceptor fluorophore, or conversely,F² may be the donor fluorophore with F¹ the acceptor fluorophore. F¹ maybe 5-carboxytetramethylrhodamine or 7-hydroxy-4-methylcoumarin-3-aceticacid, while F² may be rhodamine X acetamide or7-diethylamino-3-((4'-iodoacetyl)amino)phenyl)-4-methylcoumarin.

In a particularly preferred embodiment, n and k are 0; C¹ isAsp-Ala-Ile; P is Pro-Nle-Ser-Ile; C² is Pro-Cys; F¹ is5-carboxytetramethylrhodamine; and F² is rhodamine X acetamide. Inanother particularly preferred embodiment, n and k are 0; C¹ isAsp-Ala-Ile; P is Pro-Met-Ser-Ile; C² is Pro-Cys; F¹ is5-carboxytetramethylrhodamine; and F² is rhodamine X acetamide.

This invention also includes compositions in which C¹ is Asp-Ala-Ile; Pis Pro-Met-Ser-Ile or Pro-Nle-Ser-Ile; C² is Pro-Cys; and either n or kis 1 and either S¹ or S² is a spacer having the sequenceAsp-Gly-Ser-Gly-Gly-Gly-Glu-Asp-Glu-Lys, Lys-Glu-Asp-Gly-Gly-Asp-Lys,Asp-Gly-Ser-Gly-Glu-Asp-Glu-Lys, Asp-Gly-Gly-Gly-Lys-Lys, orLys-Glu-Asp-Glu-Gly-Ser-Gly-Asp-Lys. In these compositions F¹ may be5'-carboxytetramethylrhodamine; and F² may be rhodamine X acetamide.These compositions may be conjugated to a solid support or to a lipidincluding membrane lipids or liposomes.

In another embodiment, any of the compositions described above may beused in a method for detecting protease activity in a sample (SequenceID Nos. 26-30 respectively). The sample may be a sample of "stock"protease, such as is used in research or industry, or it may be abiological sample. Thus, this invention provides for a method ofdetecting protease activity in a sample by contacting the sample withany of the compositions described above and then detecting a change influorescence of the fluorogenic composition where an increase influorescence indicates protease activity. The sample is preferably abiological sample which may include biological fluids such as sputum orblood, tissue samples such as biopsies or sections, and cell sampleseither as biopsies or in culture. Particularly preferred are tissuesections, such as frozen sections, that have not been embedded or fixed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show an HPLC analysis of the D-NorFES-A proteaseindicator (F¹ -Asp-Ala-Ile-Pro-Nle-Ser-Ile-Pro-Cys-F² (Sequence ID No.31) protease indicator where F¹ is a donor fluorophore(5'-carboxytetramethylrhodamine (C2211) and F² is an acceptorfluorophore (rhodamine X acetamide (R492))) before and after theaddition of elastase. (FIG. 1A: HPLC before the addition of elastaseshowing the late eluting peak representing the intact indicatormolecule. FIG. 1B HPLC after the addition of elastase with detection at550 nm where both fluorophores absorb. FIG. 1C HPLC after the additionof elastase with detection at 580 nm where F² absorbs maximally.

FIGS. 2A and 2B show the emission spectra of the D-NorFES-A fluorogenicprotease indicator after (FIG. 2B) before and after the addition ofelastase.

FIG. 3 shows the time-dependent increase of the fluorogenic proteaseindicator of FIG. 1, as a function of time after addition of 1 unit ofelastase.

FIGS. 4A and 4B show the fluorescence intensity of the donor fluorophoreas a function of time after addition of 1 unit of elastase. FIG. 4A Thefluorogenic protease indicator of FIG. 1. FIG. 4B The peptide backboneof the fluorogenic protease of FIG. 1 singly labeled with each of thetwo fluorophores. D-NorFES-A is the F¹-Asp-Ala-Ile-Pro-Nle-Ser-Ile-Pro-Cys-F² protease indicator where F¹ is adonor fluorophore (5'-carboxytetramethylrhodamine (C2211) and F² is anacceptor fluorophore (rhodamine X acetamide (R492). D-NorFES andA-NorFES each designate a molecule having the same peptide backbone, butbearing only one of the two fluorophores.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The term "protease binding site" is used herein to refers to an aminoacid sequence that is characteristically recognized and cleaved by aprotease. The protease binding site contains a peptide bond that ishydrolyzed by the protease and the amino acid residues joined by thispeptide bond are said to form the cleavage site. These amino acids aredesignated P₁ and P₁ ' for the residues on the amino and carboxyl sidesof the hydrolyzed bond respectively.

A fluorophore is a molecule that absorbs light at a characteristicwavelength and then re-emits the light most typically at acharacteristic different wavelength. Fluorophores are well known tothose of skill in the art and include, but are not limited to rhodamineand rhodamine derivatives, fluorescein and fluorescein derivatives,coumarins and chelators with the lanthanide ion series. A fluorophore isdistinguished from a chromophore which absorbs, but does notcharacteristically re-emit light.

"Peptides" and "polypeptides" are chains of amino acids whose α carbonsare linked through peptide bonds formed by a condensation reactionbetween the α carbon carboxyl group of one amino acid and the aminogroup of another amino acid. The terminal amino acid at one end of thechain (amino terminal) therefore has a free amino group, while theterminal amino acid at the other end of the chain (carboxy terminal) hasa free carboxyl group. As used herein, the term "amino terminus"(abbreviated N-terminus) refers to the free α-amino group on an aminoacid at the amino terminal of a peptide or to the α-amino group (iminogroup when participating in a peptide bond) of an amino acid at anyother location within the peptide. Similarly, the term "carboxyterminus" refers to the free carboxyl group on the carboxy terminus of apeptide or the carboxyl group of an amino acid at any other locationwithin the peptide. Peptides also include peptide mimetics such as aminoacids joined by an ether as opposed to an amide bond.

The polypeptides described herein are written with the amino terminus atthe left and the carboxyl terminus at the right. The amino acidscomprising the peptide components of this invention are numbered withrespect to the protease cleavage site, with numbers increasingconsecutively with distance in both the carboxyl and amino directionfrom the cleavage site. Residues on the carboxyl site are either notatedwith a "'" as in P₁ ', or with a letter and superscript indicating theregion in which they are located. The "'" indicates that residues arelocated on the carboxyl side of the cleavage site.

The term "residue" or "amino acid" as used herein refers to an aminoacid that is incorporated into a peptide. The amino acid may be anaturally occurring amino acid and, unless otherwise limited, mayencompass known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

The term "domain" or "region" refers to a characteristic region of apolypeptide. The domain may be characterized by a particular structuralfeature such as a β turn, an alpha helix, or a β pleated sheet, bycharacteristic constituent amino acids (e.g. predominantly hydrophobicor hydrophilic amino acids, or repeating amino acid sequences), or byits localization in a particular region of the folded three dimensionalpolypeptide. As used herein, a region or domain is composed of a seriesof contiguous amino acids.

The terms "protease activity" or "activity of a protease" refer to thecleavage of a peptide by a protease. Protease activity comprises the"digestion" of one or more peptides into a larger number of smallerpeptide fragments. Protease activity of particular proteases may resultin hydrolysis at particular peptide binding sites characteristicallyrecognized by a particular protease. The particular protease may becharacterized by the production of peptide fragments bearing particularterminal amino acid residues.

The amino acids referred to herein are described by shorthanddesignations as follows:

                  TABLE 1                                                         ______________________________________                                        Amino acid nomenclature.                                                      Amino Acid Nomenclature                                                       Name               3-letter    1 letter                                       ______________________________________                                        Alanine            Ala         A                                              Arginine           Arg         R                                              Asparagine         Asn         N                                              Aspartic Acid      Asp         D                                              Cysteine           Cys         C                                              Glutamic Acid      Glu         E                                              Glutamine          Gln         Q                                              Glycine            Gly         G                                              Histidine          His         H                                              Homoserine         Hse         --                                             Isoleucine         Ile         I                                              Leucine            Leu         L                                              Lysine             Lys         K                                              Methionine         Met         M                                              Methionine sulfoxide                                                                             Met (O)     --                                             Methionine methylsulfonium                                                                       Met (S-Me)  --                                             Norleucine         Nle         --                                             Phenylalanine      Phe         F                                              Proline            Pro         P                                              Serine             Ser         S                                              Threonine          Thr         T                                              Tryptophan         Trp         W                                              Tyrosine           Tyr         Y                                              Valine             Val         V                                              α-aminoisobutyric acid                                                                     Aib                                                        ______________________________________                                    

Fluorogenic Indicators of Protease Activity

This invention provides for novel fluorogenic molecules useful fordetecting protease activity in a sample. The fluorogenic proteaseindicators of the present invention generally comprise a fluorophore(donor) linked to an "acceptor" molecule by a peptide having an aminoacid sequence that is recognized and cleaved by a particular protease.The donor fluorophore typically is excited by incident radiation at aparticular wavelength which it then re-emits at a different (longer)wavelength. When the donor fluorophore is held in close proximity to theacceptor molecule, the acceptor absorbs the light re-emitted by thefluorophore thereby quenching the fluorescence signal of the donormolecule. Cleavage of the peptide joining the donor fluorophore and theacceptor results in separation of the two molecules, release of thequenching effect and increase in fluorescence.

In one basic application, the fluorogenic molecules of this inventionmay be used to assay the activity of purified protease made up as areagent (e.g. in a buffer solution) for experimental or industrial use.Like many other enzymes, proteases may loose activity over time,especially when they are stored as their active forms. In addition, manyproteases exist naturally in an inactive precursor form (e.g. a zymogen)which itself must be activated by hydrolysis of a particular peptidebond to produce the active form of the enzyme prior to use. Because thedegree of activation is variable and because proteases may looseactivity over time, it is often desirable to verify that the protease isactive and to often quantify the activity before using a particularprotease in a particular application.

Previous approaches to verifying or quantifying protease activityinvolve combining an aliquot of the protease with its substrate,allowing a period of time for digestion to occur and then measuring theamount of digested protein, most typically by HPLC. This approach istime consuming, utilizes expensive reagents, requires a number of stepsand entails a considerable amount of labor. In contrast, the fluorogenicreagents of the present invention allow rapid determination of proteaseactivity in a matter of minutes in a single-step procedure. An aliquotof the protease to be tested is simply added to, or contacted with, thefluorogenic reagents of this invention and the subsequent change influorescence is monitored (e.g., using a fluorimeter).

In addition to determining protease activity in "reagent" solutions, thefluorogenic compositions of the present invention may be utilized todetect protease activity in biological samples. The term "biologicalsample", as used herein, refers to a sample obtained from an organism orfrom components (e.g., cells) of an organism. The sample may be of anybiological tissue or fluid. Frequently the sample will be a "clinicalsample" which is a sample derived from a patient. Such samples include,but are not limited to, sputum, blood, blood cells (e.g., white cells),tissue or fine needle biopsy samples, urine, peritoneal fluid, andpleural fluid, or cells therefrom. Biological samples may also includesections of tissues such as frozen sections taken for histologicalpurposes.

Previously described fluorogenic protease indicators typically absorblight in the ultraviolet range (e.g., Wang et at., supra. ). They arethus unsuitable for sensitive detection of protease activity inbiological samples which typically contain constituents (e.g., proteins)that absorb in the ultraviolet range. In contrast, the fluorescentindicators of the present invention both absorb and emit in the visiblerange (400 nm to about 700 nm). These signals are therefore not readilyquenched by, nor is activation of the fluorophores, that is absorbtionof light, interfered with by background molecules; therefore they areeasily detected in biological samples.

In addition, unlike previous fluorogenic protease indicators which oftenutilize a fluorophore and a quenching chromophore, the indicators of thepresent invention may utilize two fluorophores (i.e. fluorophore as bothdonor and acceptor). Pairs of fluorophores may be selected that show amuch higher degree of quenching than previously describedchromophore/fluorophore combinations. In fact, previous compositionshave been limited to relatively low efficiency fluorophores because ofthe small degree of quenching obtainable with the matching chromophore (Wang et al. supra.). In contrast, the fluorogenic protease indicators ofthis invention utilize high efficiency fluorophores and are able toachieve a high degree of quenching while providing a strong signal whenthe quench is released by cleavage of the peptide substrate. The highsignal allows detection of very low levels of protease activity. Thusthe fluorogenic protease indicators of this invention are particularlywell suited for in situ detection of protease activity.

The fluorogenic protease indicators of the present have the generalformula: ##STR4## where P is a peptide comprising a protease bindingsite, F¹ and F² are fluorophores, C¹ and C² are conformation determiningregions, and S¹ and S² are optional peptide spacers. F¹ may be the donorfluorophore while F² is the acceptor fluorophore, or conversely, F² maybe the donor fluorophore while F¹ is the acceptor fluorophore. Theprotease binding site provides an amino acid sequence (a peptide) thatis recognized and cleaved by the protease whose activity the indicatoris designed to reveal. The protease binding site is typically a straightchain peptide ranging in length from about 2 to about 8, more preferablyfrom about 2 to about 6 and most preferably 2 to about 4 amino acids inlength.

The conformation determining region is an amino acid sequence thatintroduces a bend into the molecule. The combined effect of the twoconformation determining regions is to put two bends into the peptidebackbone of the composition so that it achieves a substantially U-shapedconformation. This U-shaped conformation brings the fluorophoresattached to the amino and carboxyl termini of C¹ and C² respectivelyinto closer proximity to each other. The fluorophores are thuspreferably positioned adjacent to each other at a distance less thanabout 100 angstroms. The fluorophores (F¹ and F²) are typicallyconjugated directly to the conformation determining regions, althoughthey may be joined by linkers. The optional spacers (S¹ and S²) whenpresent, are used to link the composition to a solid support.

The substantially U-shaped conformation increases the proteasespecificity of the composition. The amino acid sequences comprising theconformation determining regions (the arms of the U) are less accessibleto the enzyme due to steric hinderance with each other and with theattached fluorophores. The protease binding site (presented on thebottom portion of the U) is relatively unobstructed by either thefluorophore or the complementarity determining region and is thusreadily accessible to the protease.

Protease Binding Site and Conformation Determining Regions

The protease binding site and conformation determining regions form acontiguous amino acid sequence (peptide). The protease binding site isan amino acid sequence that is recognized and cleaved by a particularprotease. It is well known that various proteases cleave peptide bondsadjacent to particular amino acids. Thus, for example, trypsin cleavespeptide bonds following basic amino acids such as arginine and lysineand chymotrypsin cleaves peptide bonds following large hydrophobic aminoacid residues such as tryptophan, phenylalanine, tyrosine and leucine.The serine protease elastase cleaves peptide bonds following smallhydrophobic residues such as alanine.

A particular protease, however, will not cleave every bond in a proteinthat has the correct adjacent amino acid. Rather, the proteases arespecific to particular amino acid sequences which serve as recognitiondomains for each particular protease.

Any amino acid sequence that comprises a recognition domain and can thusbe recognized and cleaved by a protease is suitable for the "proteasebinding site" of the fluorogenic protease indicator compositions of thisinvention. Known protease substrate sequences and peptide inhibitors ofproteases posses amino acid sequences that are recognized by thespecific protease they are cleaved by or that they inhibit. Thus knownsubstrate and inhibitor sequences provide the basic sequences suitablefor use in the protease recognition region. A number of proteasesubstrates and inhibitor sequences suitable for use as protease bindingdomains in the compositions of this invention are indicated in Table 2.One of skill will appreciate that this is not a complete list and thatother protease substrates or inhibitor sequences may be used.

The amino acid residues comprising the protease binding site are, byconvention, numbered relative to the peptide bond hydrolyzed by aparticular protease. Thus the first amino acid residue on the amino sideof the cleaved peptide bond is designated P₁ while the first amino acidresidue on the carboxyl side of the cleaved peptide bond is designatedP₁ '. The numbering of the residues increases with distance away fromthe hydrolyzed peptide bond. Thus a four amino acid protease bindingregion would contain amino acids designated:

    P.sub.2 -P.sub.1 -P.sub.1 '-P.sub.2 '

and the protease would cleave the binding region between P₁ and P₁ '.

In a preferred embodiment, the protease binding region of thefluorogenic protease indicators of the present invention is selected tobe symmetric about the cleavage site. Thus, for example, where a bindingregion is

    Ile-Pro-Met-Ser-ILe

(e.g. α-1 anti-trypsin, Sequence ID No. 32) and the cleavage occursbetween Met and Ser then a 4 amino acid residue binding region based onthis sequence would be: ##STR5## Other examples of binding domainsselected out of longer sequences are provided in Table 2. The remainingamino or carboxyl residues that are not within the protease bindingdomain may remain as part of the conformation determining regionssubject to certain limitations as will be explained below. Thus, in theinstant example, the amino terminal Ile may be incorporated into the C¹conformation determining region.

Various amino acid substitutions may be made to the amino acidscomprising the protease binding domain to increase binding specificity,to eliminate reactive side chains, or to increase rigidity (decreasedegrees of freedom) of the molecule. Thus, for example, it is oftendesirable to substitute methionine (Met) residues, which bear aoxidizable sulfur, with norleucine. Thus, in the example given, apreferred protease binding region will have the sequence: ##STR6##Conformation Determining Regions

Conformation determining regions (C¹ and C²) are peptide regions oneither end of the protease cleavage region that both stiffen andintroduce bends into the peptide backbone of the fluorogenic proteaseindicator molecules of this invention. The combination of the twoconformation determining regions and the relatively straight proteasecleavage region produces a roughly U-shaped molecule with the cleavagesite at the base (middle) of the "U".

Amino acids such as proline (Pro) and α-aminobutyric acid (Aib) areselected both to introduce bends into the peptide molecule and toincrease the rigidity of the peptide backbone. The C¹ and C² domains areselected such that the "arms" of the U are rigid and the attachedfluorophores are localized adjacent to each other at a separation ofless than about 100 angstroms. In order to maintain the requisitestiffness of the peptide backbone and placement of the fluorophores, theconformation determining regions are preferably 4 amino acids in lengthor less, or alternatively are greater than about 18 amino acids inlength and form a stable alpha helix conformation or a β-pleated sheet.

In a preferred embodiment, the peptide backbone of the fluorogenicprotease indicators of the present invention will comprise a tripeptideC¹ region, a tetrapeptide P region and a single amino acid or dipeptideC² region. These compounds may be represented by the formula: ##STR7##where Y is either ##STR8## In these formulas the peptide binding regionis designated -P₂ -P₁ -P₁ '-P₂ '-, while the amino acid residues ofconformation determining regions C¹ and C² are designated -C¹ ₅ -C¹ ₄-C¹ ₃ - and -C² ₃ -C² ₄ - respectively. The C² region may either be anamino acid or a dipeptide. Whether the C² region is a dipeptide or anamino acid, the F² fluorophore and the L² spacer, when present, arealways coupled to the carboxyl terminal residue of C². When a spacer ispresent on the C² region, it is attached the carboxyl terminal residueof C² by a peptide bond to the α carboxyl group.

As indicated above, the conformation determining regions typicallycontain amino acid residues such as a proline (Pro) that introduce abend into the molecule and increase its stiffness. One of skill in theart will appreciate, however that where the terminal residues of theprotease binding region (P) are themselves bend-creating residues suchas proline, it is not necessary to locate a bend-creating residue at theposition closest to P in the C region attached to that terminus. Theconformation determining regions are thus designed by first determiningthe protease binding region, as described above, determining the"left-over" residues that would lie in the conformation determiningregions, and if necessary, modifying those residues according to thefollowing guidelines:

1. If the P₂ ' site is not a proline then C² is a dipeptide (FormulaIII) Pro-Cys or Aib-Cys, while conversely, if the P₂ ' site is a prolinethen C² is an amino acid residue (Formula IV) Cys.

2. If the P₂ site is not a proline then C¹ is a tripeptide consisting ofAsp-C¹ ₄ -Pro, Asp-C¹ ₄ -Aib, Asp-Aib-Pro, Asp-Pro-C¹ ₃, Asp-Pro-Aib, orAsp-Aib-Aib, while if the P₂ site is a proline residue then group C¹ isa tripeptide consisting of Asp-C¹ ₄ -C¹ ₃ or Aib-C¹ ₄ -C¹ ₃.

3. If the P₃ residue is a proline then C¹ is a tripepride consisting ofAsp-C¹ ₄ -Pro or Asp-Aib-Pro.

4. If the P₄ residue is a proline then C¹ is a tripepride consisting ofAsp-Pro-C¹ ₃ or Asp-Pro-Aib.

5. If P₂ and C¹ ₃ are both not prolines then C¹ is a tripeptideconsisting of Asp-Pro-C¹ ₃, Asp-Aib-C¹ ₃, Asp-C¹ ₄ -Pro, Asp-C¹ ₄ -Aib,Asp-Pro-Aib, or Asp-Aib-Pro.

As indicated above, any methionine (Met) may be replaced with anorleucine (Nle). A number of suitable peptide backbones consisting ofC¹, P and C² are provided in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Illustration of the design of the conformation determining regions and        protease binding site                                                         based on known protease substrate and inhibitor sequences. Italics            indicate residues that                                                        are added to create a bend and to increase rigidity of the conformatin        determining regions.                                                          Normal font indicates residues of the substrate or inhibitor that forms       the protease binding                                                          site. Brackets indicate residues that must are preferably replaced. The       thick line indicates                                                          the location at which the protease binding site is cleaved.                             CDR (C.sup.1)                                                                           Protease Binding Site (P)                                                                  CDR (C.sup.2)                                                                       Seq                                    Substrate/Inhibitor                                                                     C.sup.1.sub.5                                                                    C.sup.1.sub.4                                                                    C.sup.1.sub.3                                                                     P.sub.2                                                                          P.sub.1                                                                          P.sub.1 '                                                                         P.sub.2 '                                                                        C.sup.2.sub.3                                                                    C.sup.2.sub.4                                                                    ID                                     __________________________________________________________________________    α-1 anti-trypsin                                                                  Asp                                                                              Ala                                                                              Ile Pro                                                                              Met                                                                              Ser Ile                                                                              Pro                                                                              Cys                                                                              35                                                            Nle       Aib                                          plasminogen                                                                             Asp                                                                              Met                                                                              (Thr)                                                                             Gly                                                                              Arg                                                                              Thr Gly                                                                              Pro                                                                              Cys                                                                              36                                     activator inhibitor                                                                        Aib                                                                              Aib              Aib                                          2            Pro                                                                              Pro                                                           neutrophil                                                                              Asp                                                                              Ala                                                                              (Thr)                                                                             Phe                                                                              Cys                                                                              Met Leu                                                                              Pro                                                                              Cys                                                                              37                                     leukocyte elastase                                                                         Aib                                                                              Aib       Nle    Aib                                          inhibitor       Pro                                                           anti-plasmin                                                                            Asp                                                                              Aib                                                                              (Ser)                                                                             Arg                                                                              Met                                                                              Ser Leu                                                                              Pro                                                                              Cys                                                                              38                                     inhibitor       Aib    Nle       Aib                                                          Pro                                                           anti-α-1 thrombin                                                                 Asp                                                                              Ile                                                                              (Ala)                                                                             Gly                                                                              Arg                                                                              Ser Leu                                                                              Pro                                                                              Cys                                                                              39                                                  Aib                                                                              Aib              Aib                                                          Pro                                                           α-1 Asp                                                                              Aib                                                                              (Thr)                                                                             Leu                                                                              Leu                                                                              Ser Leu                                                                              Pro                                                                              Cys                                                                              40                                     antichymotrypsin                                                                              Aib              Aib                                                          Pro                                                           interstitial type                                                                       Asp                                                                              Gly                                                                              Pro Leu                                                                              Gly                                                                              Ile Ala                                                                              Pro                                                                              Cys                                                                              41                                     III (human liver)                                                                          Aib                                                                              Aib              Aib                                          collagen                                                                      type I collagen for                                                                     Asp                                                                              Gly                                                                              Pro Gln                                                                              Gly                                                                              Ile Leu                                                                              Pro                                                                              Cys                                                                              42                                     collagenase Bovine                                                                         Aib                                                                              Aib              Aib                                          α 1    Pro                                                              type I collagen                                                                         Asp                                                                              Gly                                                                              Pro Gln                                                                              Gly                                                                              Leu Leu                                                                              Pro                                                                              Cys                                                                              43                                     chick α2                                                                             Aib                                                                              Aib              Aib                                                       Pro                                                              human α1 type II                                                                  Asp                                                                              Gly                                                                              Pro Gln                                                                              Gly                                                                              Ile Ala                                                                              Pro                                                                              Cys                                                                              44                                     collagen     Aib                                                                              Aib              Aib                                                       Pro                                                              type III collagen -                                                                     Asp                                                                              Gly                                                                              Pro Gln                                                                              Ala                                                                              Ile Ala                                                                              Pro                                                                              Cys                                                                              45                                     AIA          Aib                                                                              Aib              Aib                                                       Pro                                                              type III collagen                                                                       Asp                                                                              Gly                                                                              Pro Gln                                                                              Gly                                                                              Ile Ala                                                                              Pro                                                                              Cys                                                                              55                                     (human skin) Aib                                                                              Aib              Aib                                                       Pro                                                              human α 2                                                                         Asp                                                                              Gly                                                                              Pro Glu                                                                              Gly                                                                              Leu Arg                                                                              Pro                                                                              Cys                                                                              46                                     macroglobulin                                                                              Aib                                                                              Aib              Aib                                                       Pro                                                              stromelysin                                                                             Asp                                                                              Asp                                                                              (Val)                                                                             Gly                                                                              His                                                                              Phe Arg                                                                              Pro                                                                              Cys                                                                              47                                     cleavage sites of                                                                          Aib                                                                              Aib              Aib                                          stromelysin-1d                                                                             Pro                                                                              Pro                                                           stromelysin                                                                             Asp                                                                              Asp                                                                              (Thr)                                                                             Leu                                                                              Glu                                                                              Val Met                                                                              Pro                                                                              Cys                                                                              48                                     cleavage sites of                                                                          Aib                                                                              Aib           Nle                                                                              Aib                                          stromelysin-1                                                                              Pro                                                                              Pro                                                           stromelysin                                                                             Asp                                                                              Arg                                                                              (Ala)                                                                             Ile                                                                              His                                                                              Ile Gln                                                                              Pro                                                                              Cys                                                                              49                                     cleavage site of                                                                           Aib                                                                              Aib              Aib                                          proteoglycan link                                                                          Pro                                                                              Pro                                                           protein                                                                       gelatinase type IV                                                                      Asp                                                                              Asp                                                                              (Val)                                                                             Ala                                                                              Asn                                                                              Tyr Asn                                                                              Pro                                                                              Cys                                                                              56                                     collagenase site of                                                                        Aib                                                                              Aib              Aib                                          72 K gelatinases                                                                           Pro                                                                              Pro                                                           gelatinase type IV                                                                      Asp                                                                              Gly                                                                              Pro Ala                                                                              Gly                                                                              Glu Arg                                                                              Pro                                                                              Cys                                                                              50                                     cleavage of gelatin                                                                        Aib                                                                              Aib              Aib                                                       Pro                                                              gelatinase type IV                                                                      Asp                                                                              Gly                                                                              Pro Ala                                                                              Gly                                                                              Phe Ala                                                                              Pro                                                                              Cys                                                                              51                                     cleavage of gelatin                                                                        Aib                                                                              Aib              Aib                                                       Pro                                                              type III collagen                                                                       Asp                                                                              Gly                                                                              Pro Gln                                                                              Gly                                                                              Leu Ala                                                                              Pro                                                                              Cys                                                                              52                                     (human skin) Aib                                                                              Aib              Aib                                                       Pro                                                              Human FIB-CL                                                                            Asp                                                                              Asp                                                                              (Val)                                                                             Ala                                                                              Gln                                                                              Phe Val                                                                              Pro                                                                              Cys                                                                              53                                     propeptide   Aib                                                                              Aib              Aib                                                       Pro                                                                              Pro                                                           Gelatin α1 (type 1)                                                               Asp                                                                              Gly                                                                              Pro Ala                                                                              Gly                                                                              Val Gln                                                                              Pro                                                                              Cys                                                                              54                                                  Aib                                                                              Aib              Aib                                                       Pro                                                              __________________________________________________________________________

"Donor" and "Acceptor" Fluorophores

A fluorophore excited by incident radiation absorbs light and thensubsequently re-emits that light at a different (longer) wavelength.However, in the presence of a second class of molecules, known as"acceptors" the light emitted by a so-called donor fluorophore isabsorbed by the acceptor thereby quenching the fluorescence signal ofthe donor. Thus, use of two fluorophores, as opposed to afluorophore/chomophore pair, allows a clearer assessment of the overlapbetween the emission spectrum of the donor and the excitation spectrumof the acceptor. This facilitates the design of a peptide backbone thatallows allowing optimization of the quenching. This results in a highefficiency donor/acceptor pair facilitating the detection of lowconcentrations of protease activity. Thus, although afluorophore/chromophore combination may be suitable, in a preferredembodiment, the fluorogenic protease inhibitors of this invention willcomprise two fluorophores.

The "donor" and "acceptor" molecules are typically selected as a matchedpair such that the absorption spectra of the acceptor molecule overlapsthe emission spectrum of the donor molecule as much as possible. Inaddition, the donor and acceptor fluorophores are preferably selectedsuch that both the absorbtion and the emission spectrum of the donormolecule is in the visible range (400 nm to about 700 nm). Thefluorophores thereby provide a signal that is detectable in a biologicalsample thus facilitating the detection of protease activity inbiological fluids, tissue homogenates, in situ in tissue sections, andthe like. The emission spectra, absorption spectra and chemicalcomposition of many fluorophores are well known to those of skill in theart (see, for example, Handbook of Fluorescent Probes and ResearchChemicals, R. P. Haugland, ed. which is incorporated herein byreference).

Preferred fluorophore pairs include the rhodamine derivatives. Thus, forexample 5-carboxytetramethylrhodamine (C2211 available from MolecularProbes, Eugene, Wash., U.S.A.) (Formula V) is a particularly preferreddonor molecule and ##STR9## rhodamine X acetamide (R492 from MolecularProbes) (Formula VI) is a particularly preferred receptor molecule.These fluorophores are particularly preferred since the ##STR10##excitation and emission of both members of this donor/acceptor pair arein the visible wavelengths, the molecules have high extinctioncoefficients and the molecules have high fluorescence yields insolution. The extinction coefficient is a measure of the lightabsorbance at a particular wavelength by the chromophore and istherefore related to its ability to quench a signal, while thefluorescence yield is the ratio of light absorbed to light re-emittedand is a measure of the efficiency of the fluorophore and thus effectsthe sensitivity of the protease indicator.

Of course, while not most preferred, fluorophores that absorb and emitin the ultraviolet may also be used in the protease indicators of thepresent invention. One particularly preferred ultraviolet absorbing pairof fluorophores is 7-hydroxy-4-methylcoumarin-3-acetic acid as the donormolecule (Formula VII) ##STR11## and7-diethylamino-3-((4'-iodoacetyl)amino)phenyl)-4-methylcoumarin (FormulaVIII) as the acceptor molecule. ##STR12## These and other fluorophoresare commercially available from a large number of manufacturers such asMolecular Probes (Eugene, Oreg., U.S.A.).

Preparation of Fluorogenic Protease Indicators

The fluorogenic protease indicators of the present invention arepreferably prepared by first synthesizing the peptide backbone, i.e. theprotease cleavage site (P), the two conformation determining regions (C¹and C²), and the spacers (S¹ and S²) if present. The fluorophores arethen chemically conjugated to the peptide. The fluorophores arepreferably conjugated directly to the peptide however, they may also becoupled to the peptide through a linker. Finally, where the fluorogenicprotease indicator is to be bound to a solid support, it is thenchemically conjugated to the solid support via the spacer (S¹ or S²)either directly or through a linker.

a) Preparation of the Peptide Backbone

Solid phase peptide synthesis in which the C-terminal amino acid of thesequence is attached to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence is the preferredmethod for preparing the peptide backbone of the compounds of thepresent invention. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc. 85,2149-2156 (1963), and Gross and Meienhofer, eds. Academic press, N.Y.,1980 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill. (1984) which are incorporated herein byreference. Solid phase synthesis is most easily accomplished withcommercially available peptide synthesizers utilizing FMOC or TBOCchemistry. The chemical synthesis of the peptide component of afluorogenic protease indicator is described in detail in Example 1.

Alternatively, the peptide components of the fluorogenic proteaseindicators of the present invention may be synthesized utilizingrecombinant DNA technology. Briefly, a DNA molecule encoding the desiredamino acid sequence is synthesized chemically by a variety of methodsknown to those of skill in the art including the solid phasephosphoramidite method described by Beaucage and Carruthers, Tetra.Letts. 22:1859-1862 (1981), the triester method according to Matteueci,et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein byreference, or by other methods known to those of skill in the art. It ispreferred that the DNA be synthesized using standard β-cyanoethylphosphoramidites on a commercially available DNA synthesizer usingstandard protocols.

The oligonucleotides may be purified, if necessary, by techniques wellknown to those of skill in the art. Typical purification methodsinclude, but are not limited to gel electrophoresis, anion exchangechromatography (e.g. Mono-Q column, Pharmacia-LKB, Piscataway, N.J.,U.S.A.), or reverse phase high performance liquid chromatography (HPLC).Method of protein and peptide purification are well known to those ofskill in the art. For a review of standard techniques see, Methods inEnzymology Volume 182: Guide to Protein Purification, M. Deutscher, ed.(1990), pages 619-626, which are incorporated herein by reference.

The oligonucleotides may be converted into double stranded DNA either byannealing with a complementary oligonucleotide or by polymerization witha DNA polymerase. The DNA may then be inserted into a vector under thecontrol of a promoter and used to transform a host cell so that the cellexpresses the encoded peptide sequence. Methods of cloning andexpression of peptides are well known to those of skill in the art. See,for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory (1989)), Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques (Berger andKimmel (eds.), San Diego: Academic Press, Inc. (1987)), or CurrentProtocols in Molecular Biology, (Ausubel, et al. (eds.), GreenePublishing and Wiley-Interscience, New York (1987), which areincorporated herein by reference.

b) Linkage of the Fluorophores to the Peptide Backbone

The fluorophores are linked to the peptide backbone by any of a numberof means well known to those of skill in the art. In a preferredembodiment, the fluorophore is linked directly from a reactive site onthe fluorophore to a reactive group on the peptide such as a terminalamino or carboxyl group, or to a reactive group on an amino acid sidechain such as a sulfur, an amino, or a carboxyl moiety. Manyfluorophores normally contain suitable reactive sites. Alternatively,the fluorophores may be derivatized to provide reactive sites forlinkage to another molecule. Fluorophores derivatized with functionalgroups for coupling-to a second molecule are commercially available froma variety of manufacturers. The derivatization may be by a simplesubstitution of a group on the fluorophore itself, or may be byconjugation to a linker. Various linkers are well known to those ofskill in the art and are discussed below.

As indicated above, in a preferred embodiment, the fluorophores aredirectly linked to the peptide backbone of the protease indicator. Thus,for example, the 5'-carboxytetramethylrhodamine (C2211 ) fluorophore maybe linked to aspartic acid via the alpha amino group of the amino acidas shown in Formula V. The iodoacetamide group of rhodamine X acetamide(R492)) may be linked by reaction with the sulfhydryl group of a cysteinas indicated in formula VI. Means of performing such couplings are wellknown to those of skill in the art, and the details of one such couplingare provided in Example 1.

One of skill in the art will appreciate that when the peptide spacers(S¹ or S²) are present (as is discussed below), the fluorophores arepreferably linked to the conformation determining regions through areactive group on the side chain of the terminal amino acid of C¹ or C²as the spacers themselves form a peptide linkage with the terminal aminoand carboxyl groups of C¹ or C² respectively.

c) Selection of Spacer Peptides and Linkage to a Solid Support

The fluorogenic protease indicators of the present invention may beobtained in solution or linked to a solid support. A "solid support"refers to any solid material that does not dissolve in or react with anyof the components present in the solutions utilized for assaying forprotease activity using the fluorogenic protease indicator molecules ofthe present invention and that provides a functional group forattachment of the fluorogenic molecule. Solid support materials are wellknown to those of skill in the art and include, but are not limited tosilica, controlled pore glass (CPG), polystyrene, polystyrene/latex,carboxyl modified teflon, dextran, derivatized polysaccharaides such asagar bearing amino, carboxyl or sulfhydryl groups, various plastics suchas polyethylene, acrylic, and the like. Also of use are "semi-solid"supports such as lipid membranes as found in cells and in liposomes. Oneof skill will appreciate that the solid supports may be derivatized withfunctional groups (e.g. hydroxyls, amines, carboxyls, esters, andsulfhydryls) to provide reactive sites for the attachment of linkers orthe direct attachment of the peptide.

The fluorogenic protease indicators may be linked to a solid supportdirectly through the fluorophores or through the peptide backbonecomprising the indicator. Linkage through the peptide backbone is mostpreferred.

When it is desired to link the indicator to a solid support through thepeptide backbone, the peptide backbone may comprise an additionalpeptide spacer (designated S¹ or S² in Formula I). The spacer may bepresent at either the amino or carboxyl terminus of the peptide backboneand may vary from about 1 to about 50 amino acids, more preferably from1 to about 20 and most preferably from 1 to about 10 amino acids inlength. Particularly preferred spacers includeAsp-Gly-Ser-Gly-Gly-Gly-Glu-Asp-Glu-Lys, Lys-Glu-Asp-Gly-Gly-Asp-Lys,Asp-Gly-Ser-Gly-Glu-Asp-Glu-Lys, andLys-Glu-Asp-Glu-Gly-Ser-Gly-Asp-Lys.

The amino acid composition of the peptide spacer is not critical as thespacer just serves to separate the active components of the moleculefrom the substrate thereby preventing undesired interactions. However,the amino acid composition of the spacer may be selected to provideamino acids (e.g. a cysteine) having side chains to which a linker orthe solid support itself, is easily coupled. Alternatively the linker orthe solid support itself may be attached to the amino terminus of S¹ orthe carboxyl terminus of S².

In a preferred embodiment, the peptide spacer is actually joined to thesolid support by a linker. The term "linker", as used herein, refers toa molecule that may be used to link a peptide to another molecule, (e.g.a solid support, fluorophore, etc.). A linker is a hetero orhomobifunctional molecule that provides a first reactive site capable offorming a covalent linkage with the peptide and a second reactive sitecapable of forming a covalent linkage with a reactive group on the solidsupport. The covalent linkage with the peptide (spacer) may be viaeither the terminal carboxyl or amino groups or with reactive groups onthe amino acid side-chain (e.g. through a disulfide linkage to acysteine).

Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. The linkersmay be joined to the carboxyl and amino terminal amino acids throughtheir side groups (e.g., through a disulfide linkage to cysteine).

Particularly preferred linkers are capable of forming covalent bonds toamino groups, carboxyl groups, or sulfhydryl. Amino-binding linkersinclude reactive groups such as carboxyl groups, isocyanates,isothiocyanates, esters, haloalkyls, and the like. Carboxyl-bindinglinkers are capable of forming include reactive groups such as variousamines, hydroxyls and the like. Finally, sulfhydryl-binding linkersinclude reactive groups such as sulfhydryl groups, acrylates,isothiocyanates, isocyanates and the like. Particularly preferredlinkers include sulfoMBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimideester) for linking amino groups (e.g. an amino group found on a lysineresidue in the peptide) with sulfhydryl groups found on the solidsupport, or vice versa, for linking sulfhydryl groups (e.g. found on acysteine residue of the peptide) with amino groups found on the solidsupport. Other particularly preferred linkers include EDC(1-ethyl-3-(3-dimethylaminopropryl)-carbodiimide) andbis-(sulfosuccinimidyl suberate). Other suitable linkers are well knownto those of skill in the art.

The fluorogenic compounds of the present invention may be linked to thesolid support through either the S¹ or the S² spacer such that the donorfluorophore is either retained on the solid support after cleavage ofthe molecule by a protease or such that the donor fluorophore goes intosolution after cleavage. In the former case, the substrate is thenassayed for fluorescence to detect protease activity, while in the latercase the solution is assayed for fluorescence to detect proteaseactivity.

Detection of Protease Activity

The present invention also provides methods for utilizing thefluorogenic protease indicators to detect protease activity in a varietyof contexts. Thus, in one embodiment, the present invention provides fora method of using the fluorogenic indicators to verify or quantify theprotease activity of a stock solution of a protease used forexperimental or industrial purposes. Verification of protease activityof stock protease solutions before use is generally recommended asproteases often to loose activity over time (e.g. throughself-hydrolysis) or to show varying degrees of activation when activatedfrom zymogen precursors.

Assaying for protease activity of a stock solution simply requiresadding a quantity of the stock solution to a fluorogenic proteaseindicator of the present invention and measuring the subsequent increasein fluorescence. The stock solution and the fluorogenic indicator mayalso be combined and assayed in a "digestion buffer" that optimizesactivity of the protease. Buffers suitable for assaying proteaseactivity are well known to those of skill in the art. In general, abuffer will be selected whose pH corresponds to the pH optimum of theparticular protease. For example, a buffer particularly suitable forassaying elastase activity consists of 50 mM sodium phosphate, 1 mM EDTAat pH 8.9. The measurement is most easily made in a fluorometer, andinstrument that provides an "excitation" light source for thefluorophore and then measures the light subsequently emitted at aparticular wavelength. Comparison with a control indicator solutionlacking the protease provides a measure of the protease activity. Theactivity level may be precisely quantified by generating a standardcurve for the protease/indicator combination in which the rate of changein fluorescence produced by protease solutions of known activity isdetermined.

While detection of the fluorogenic compounds is preferably accomplishedusing a fluorometer, detection may by a variety of other methods wellknown to those of skill in the art. Thus for example, since thefluorophores of the present invention emit in the visible wavelengths,detection may be simply by visual inspection of fluorescence in responseto excitation by a light source. Detection may also be by means of animage analysis system utilizing a video camera interfaced to andigitizer or other image acquisition system. Detection may also be byvisualization through a filter as under a fluorescence microscope. Themicroscope may just provide a signal that is visualized by the operator.However the signal may be recorded on photographic film or using a videoanalysis system. The signal may also simply be quantified in realtimeusing either an image analysis system or simply a photometer.

Thus, for example, a basic assay for protease activity of a sample willinvolve suspending or dissolving the sample in a buffer (at the pHoptima of the particular protease being assayed), adding to the bufferone of the fluorogenic protease indicators of the present invention, andmonitoring the resulting change in fluorescence using aspectrofluorometer. The spectrofluorometer will be set to excite thedonor fluorophore at the excitation wavelength of the donor fluorophoreand to detect the resulting fluorescence at the emission wavelength ofthe donor fluorophore.

In another embodiment, the protease activity indicators of the presentinvention may be utilized for detection of protease activity inbiological samples. Thus, in a preferred embodiment, this inventionprovides for methods of detecting protease activity in isolatedbiological samples such as sputum, blood, blood cells, tumor biopsies,and the like, or in situ, in cells or tissues in culture, or in sectionwhere the section is unimbedded and unfixed.

a) Ex vivo Assays of Isolated Biological Samples

In one embodiment, the present invention provides for methods ofdetecting protease activity in an isolated biological sample. This maybe determined by simply contacting the sample with a fluorogenicprotease indicator of the present invention and monitoring the change influorescence of the indicator over time. The sample may be suspended ina "digestion buffer" as described above. The sample may also be clearedof cellular debris, e.g. by centrifugation before analysis.

Where the fluorogenic protease indicator is bound to a solid support theassay may involve contacting the solid support bearing the indicator tothe sample solution. Where the indicator is joined to the solid supportby the side of the molecule beating the donor fluorophore, thefluorescence of the support resulting from digestion of the indicatorwill then be monitored over time by any of the means described above.Conversely, where the acceptor molecule fluorophore is bound to a solidsupport, the test solution may be passed over the solid support and thenthe resulting luminescence of the test solution (due to the cleavedfluorophore) is measured. This latter approach may be particularlysuitable for high throughput automated assays.

b) In situ Assays of Histological Sections

In another embodiment, this invention provides for a method of detectingin situ protease activity in histological sections. This method ofdetecting protease activity in tissues offers significant advantagesover prior art methods (e.g. specific stains, antibody labels, etc.)because, unlike simple labeling approaches, in situ assays using theprotease indicators indicate actual activity rather than simple presenceor absence of the protease. Proteases are often present in tissues intheir inactive precursor (zymogen) forms which are capable of bindingprotease labels. Thus traditional labeling approaches provide noinformation regarding the physiological state, visa vis proteaseactivity, of the tissue.

The in situ assay method generally comprises providing a tissue section(preferably a frozen section), contacting the section with one of thefluorogenic protease indicators of the present invention, andvisualizing the resulting fluorescence. Visualization is preferablyaccomplished utilizing a fluorescence microscope. The fluorescencemicroscope provides an "excitation" light source to induce fluorescenceof the "donor" fluorophore. The microscope is typically equipped withfilters to optimize detection of the resulting fluorescence. Thus, forexample, for the fluorogenic protease indicators described in Example 1,a typical filter cube for a Nikon microscope would contain an excitationfilter (λ=510±12 nm), a dichroic mirror (λ=580 nm) and an emissionfilter (λ=580±10 nm). As indicated above, the microscope may be equippedwith a camera, photometer, or image acquisition system.

The sections are preferably cut as frozen sections as fixation orembedding will destroy protease activity in the sample.

The fluorogenic indicator may be introduced to the sections in a numberof ways. For example, the fluorogenic protease indicator may be providedin a buffer solution, as described above, which is applied to the tissuesection. Alternatively, the fluorogenic protease indicator may beprovided as a semi-solid medium such as a gel or agar which is spreadover the tissue sample. The gel helps to hold moisture in the samplewhile providing a signal in response to protease activity. Thefluorogenic protease indicator may also be provided conjugated to apolymer such as a plastic film which may be used in procedures similarto the development of Western Blots. The plastic film is placed over thetissue sample on the slide and the fluorescence resulting from cleavedindicator molecules is viewed in the sample tissue under a microscope.

Typically the tissue sample must be incubated for a period of time toallow the endogenous proteases to cleave the fluorogenic proteaseindicators. Incubation times will range from about 10 to 60 minutes at37° C.

c) In situ Assays of Cells in Culture

In yet another embodiment, this invention provides for a method ofdetecting in situ protease activity of cells in culture. The culturedcells are grown either on chamber slides or in suspension and thentransferred to histology slides by cytocentrifugation. The slide iswashed with phosphate buffered saline and coated with a semi-solidpolymer or a solution containing the fluorogenic protease indicator. Theslide is incubated at 37° C. for the time necessary for the endogenousproteases to cleave the protease indicator. The slide is then examinedunder a fluorescence microscope equipped with the appropriate filters asdescribed above.

Protease Activity Detection Kits

The present invention also provides for kits for the detection ofprotease activity in samples. The kits comprise one or more containerscontaining the fluorogenic protease indicators of the present invention.The indicators may be provided in solution or bound to a solid support.Thus the kits may contain indicator solutions or indicator "dipsticks",blotters, culture media, and the like. The kits may also containindicator cartridges (where the fluorogenic indicator is bound to thesolid support by the "acceptor" fluorophore side) for use in automatedprotease activity detectors.

The kits additionally may include an instruction manual that teaches themethod and describes use of the components of the kit. In addition, thekits may also include other reagents, buffers, various concentrations ofprotease inhibitors, stock proteases (for generation of standard curves,etc), culture media, disposable cuvettes and the like to aid thedetection of protease activity utilizing the fluorogenic proteaseindicators of the present invention.

EXAMPLES

The invention is illustrated by the following examples. These examplesare offered by way of illustration, not by way of limitation.

Example 1

Synthesis of Fluorogenic Molecule for Detecting Protease Activity

a) Synthesis of the Peptide Backbone

The amino acid sequences Asp-Ala-Ile-Pro-Nle-Ser-Ile-Pro-Cys (SequenceID No. 31) (where C¹ is Asp-Ala-Ile, P is Pro-Nle-Ser-Ile (Sequence IDNo. 2) and C² is Pro-Cys) and Asp-Ala-Ile-Pro-Met-Ser-Ile-Pro-Cys(Sequence ID No. 32) (where C¹ is Asp-Ala-Ile, P is Pro-Met-Ser-Ile(Sequence ID No. 1) and C² is Pro-Cys) were synthesized manuallyutilizing a t-Boc Cys-Pam resin and t-Boc chemistry using the protocolfor a coupling cycle given below in Table 3. The synthesized peptideswere deprotected by treatment with hydrofluoric acid for 60 minutesunder anhydrous conditions at a temperature of 4° C.

                  TABLE 3                                                         ______________________________________                                        Reaction steps for one coupling cycle (addition of one amino acid)            for the synthesis of the peptide backbone of the                              fluorogenic protease inhibitors.                                                                         Time                                               Step Process               (min)   # Repeats                                  ______________________________________                                        1    TFA/DCM Pre-wash      2.0     1                                               15 ml, 50% (v/v) or 35% (v/v)                                            2    TFA/DCM Main Wash     28.0    1                                               15 ml, 50% (v/v) or 35% (v/v)                                            3    DCM washes, 10 ml     <0.33   4                                          4    DIEA/DCM, 10% (v/v), 10 ml                                                                          2.0     1                                          5    DIEA/DCM, 10% (v/v), 10 ml                                                                          8.0     1                                          6    DCM washes, 10 ml     <0.33   5                                          7    Resin wash with NMP (or DMF)                                                                        <0.33   5                                          8    5-fold excess t-Boc-amino acid with                                                                 90 to   1                                               HOBT and DIPC in NMP or DMF                                                                         120                                                9    DCM wash              <0.33   1                                          10   MeOH wash             <0.33   1                                          11   DCM washes            <0.33   2                                          12   DIEA wash             <0.33   1                                          13   DCM washes            <0.33   3                                          14   Ninhydrin             5.0     1                                          15   3-fold excess t-Boc-amino                                                                           30      1                                               acid/DIPC/DCM                                                            16   MeOH wash             <0.33   1                                          17   DCM washes            <0.33   5                                          18   Ninhydrin test        5       1                                          19   Go to step 1 to couple next amino                                             acid                                                                     ______________________________________                                    

Crude post-HF deprotected and cleaved peptides were purified by reversephase HPLC using a preparative C₁₈ column (YMC, Inc., Chariestown, N.C.,U.S.A.). The solvent system utilized was water and acetonitrilecontaining 0.1% (v/v) trifluoroacetic acid CFFA). HPLC was performedusing the following gradient at a flow rate of 10 ml/minute:

                  TABLE 4                                                         ______________________________________                                        HPLC gradient for purification of the peptide backbone.                       Time       % Solvent A                                                        (min)      (H.sub.2 O with TFA (0.1%)                                         ______________________________________                                        0          100                                                                7          100                                                                8          90                                                                 68         50                                                                 78         50                                                                 ______________________________________                                    

Purification of methionine-containing peptides required subjecting thepeptide to reducing conditions, e.g., dithiothreitol (DTT) and heat, toreduce methionine oxide to methionine. This reductive treatment wascarried out by dissolving the peptide in 150 mM sodium phosphate bufferwith 1 mM DTT, pH 7.5 for 30 minutes at 60° to 80° C. The reductioncould also have been carded out under a weak acidic pH with 0.03N HCLbut would have required a longer heating time. The subsequently HPLCpurified methionine containing peptides were found to oxidize uponsitting either in aqueous solution or lyophilized form. The oxidizedpeptides were found to be reducible repeating the above reducingcondition.

b) Derivatization of the Peptide Backbone With the Fluorophore Molecules

The peptides were derivatized sequentially with donor and acceptorfluorophores. Specifically the donor fluorophore(5'-carboxytetramethylrhodamine (C2211), available from MolecularProbes, Inc. Eugene, Oreg., U.S.A.) was first covalently linked to theamino terminus of the peptide. The peptide and the probe, at a molarratio of 3:1 peptide to probe, were dissolved in a minimal amount ofsolvent NMP (N-methyl pyrolidone), usually 20 to 60 ml. One molarequivalent of DIEA (diisopropyl ethyl amine) was also added to thereaction mixture. The reaction was then incubated at 37° C. with timesranging between 12 hours and 3 days. After two days an additional 1molar equivalent of dye molecule was sometimes added. The derivatizationreached nearly maximal yield by 3 days. The peptide bearing the singleC2211 fluorophore was then HPLC purified as described below.

The second (acceptor) fluorophore (rhodamine X acetamide (R492)) wasthen coupled to the carboxyl cystein of the peptide by a linkage betweenthe iodoacetamide group of the fluorophore and the sulfhydryl group ofthe terminal cystein. This coupling was accomplished as described abovefor the first fluorophore.

The complete fluorogenic protease indicator was then purified by HPLCusing an analytical reverse phase C₃ columns (2 ml void volume) fromWaters Associates Inc. (Milford, Mass., U.S.A.) using the gradient shownin Table 5 running at 1 ml/minute.

                  TABLE 5                                                         ______________________________________                                        HPLC gradient for purification of the peptide bearing                         fluorophores.                                                                 Time       % Solvent A                                                        (min)      (H.sub.2 O with TFA (0.1%)                                         ______________________________________                                        0          100                                                                1          100                                                                2          80                                                                 6          80                                                                 66         50                                                                 ______________________________________                                    

The slow reactivity of both the amino and sulfhydryl groups in couplingthe fluorophores appeared to be a function of the folded structure ofthe peptide backbone which sterically hindered access to the peptide'sreactive groups. Control experiments using irrelevant linear peptidesshowed considerably faster linking. The folded structure is alsosupported by the results reported in Examples 2 and 3. A computer energyminimization model of the peptide also indicated possible preference forthe peptide to assume a folded structure rather than an open extendedstructure. This is due largely to the presence of the conformationdetermining regions containing the two proline residues.

Example 2

The Fluorogenic Protease Indicators Provide a Strong Signal WhenDigested

In order to demonstrate that the fluorogenic protease indicators of thisinvention are easily digested by a protease, the degree of cleavage wasdetermined by assaying for the appearance of indicator cleavage productsin the presence of a protease.

Approximately 1 microgram of protease indicator, having the formula F¹-Asp-Ala-Ile-Pro-Nle-Ser-Ile-Pro-Cys-F² where F¹ is a donor fluorophore(5'-carboxytetramethylrhodamine (C2211)) linked to aspartic acid via thealpha amino group and F² is an acceptor fluorophore (rhodamine Xacetamide (R492)) linked via the sulfhydryl group of the cystein wasdissolved in a buffer consisting of 50 mM sodium phosphate, 1 mM EDTA atpH 8.9. To this solution was added 1 unit of elastase. The solution wasanalyzed by HPLC before and about 30 minutes after the addition ofelastase. The digestion was carded out at 37° C. The HPLC separatedcomponents were monitored at a wavelength of 550 nm which alloweddetection of both the C2211 fluorophore the R492 fluorophore and at 580nm which allowed detection of the R492 fluorophore.

The results are indicated in FIG. 1 which shows the HPLC profiles of thefluorogenic protease indicator solution before and after addition of theprotease elastase. FIG. 1(a) shows the HPLC before addition of theelastase showing a single peak representing the intact fluorogenicprotease inhibitor. After addition of the elastase (FIGS. 1(b) and 1(c))there was no trace of the late eluting single peak (FIG. 1(a))indicating complete digestion of the fluorogenic protease indicator. Inaddition, the two predominant peaks in FIGS. 1(b) and 1(c) indicate thatthe digestion occurred primarily at a single site. There are a fewsmaller peaks indicating a low degree of digestion at other sites withinthe peptide sequence, however, the striking predominance of only twodigestion peaks suggests that these secondary sites were not readilyaccessible to the elastase.

Changes in the emission spectrum of the fluorogenic protease indicatorafter the addition of an elastase protease was monitored using an SLMspectrofluorometer model 48000 with slit widths set at 4 nm on both theexcitation and emission sides. All measurements were carded out at 37°C.

Spectra in FIG. 2 show emission of the fluorogenic protease indicator(a) before and (b) after addition of elastase, while the time dependentincrease of the indicator's donor fluorophore emission intensity, afteraddition of elastase, is plotted in FIG. 3. The fluorogenic proteaseinhibitor showed more than a 10 fold increase in fluorescence at 589 nmafter treatment with the elastase protease (FIG. 2(a) compared to FIG.2(b)) with over a 5 fold increase in fluorescence occurring within thefirst 1000 seconds of exposure to the protease. The changes in intensitybetween treated and untreated indicators are, to some degree, a functionof slit widths used, since they represent the signal integrated acrossthe particular slit width. Thus, if wider slit widths were used (e.g. 8or 16 nm slits) an even greater signal would be provided in response todigestion.

Example 3

The Fluorescence Signal Was Due to lntramolecular Energy Dequenching

In order to show that the fluorescence increase observed after proteasetreatment was due to intramolecular energy dequenching, the signalproduced by elastase digestion of the fluorogenic protease indicator wascompared to the signal produced by elastase treatment of the samepeptide backbone coupled to either F¹ (C2211) or to F² (R492). Thechange in fluorescence intensity of the donor fluorophore after additionof 1 unit of elastase to equal concentrations of the double-fluorophoremolecule and the two single-fluorophore molecules.

The results are illustrated in FIG. 4. The double-fluorophore moleculeshowed nearly complete quenching initially, followed by a dramaticincrease in fluorescence after addition of the elastase which reached aconstant value approximately 30 minutes after addition of the elastase(FIG. 4(a)). In contrast, the two single-fluorophore molecules showedvirtually no initial quenching and no significant change in fluorescenceafter addition of the elastase. In fact, the fluorescence level wascomparable to the fluorescence level of the fully digesteddouble-fluorophore indicator molecule (FIG. 4(b)).

These results indicate that the increase in fluorescence intensity ofthe fluorogenic protease indicator is due to interruption of theresonance energy transferred intramolecularly from the donor fluorophoreto the acceptor fluorophore and not to interaction between thefluorophore and the peptide backbone. This is significant since it isknown that upon binding to a large protein or hydrophobic peptide thefluorescence of many hydrophobic fluorophores is quenched.

Example 4

Without being bound to a particular theory, it is believed that thefluorogenic protease indicators of the present invention achieve a highdegree of protease specificity due to their folded structure, moreparticularly due to their relative rigid U-shaped conformation. Thefluorescence obtained from the molecule reflects the average separationof two fluorophores. Thus, it was predicted that if the proteaseindicators existed in a relatively unfolded or flexible state,conditions that tend to cause unfolding (denaturation) would have littleor no effect on the fluorescence of the molecule in the absence of aprotease. Conversely, if the molecule is relatively rigid, thendenaturing conditions would be expected to increase the fluorescencesignal as the average separation of the fluorophores would be expectedto increase thereby decreasing the quenching effect.

Thus, the effect of denaturing conditions on the fluorescence of thefluorogenic protease indicator in the absence of a protease wasdetermined. First the change of fluorescence of the indicator of Example1, as a function of temperature, was measured. Relative fluorescenceintensity (of the donor fluorophore) increased approximately linearlyfrom a value of about 0.6 (relative fluorescence units (RUs)) at 25°° C.to a value of about 1.2 RUs at 50° to 55° C. and thereafter reached aplateau.

Similarly when denatured with a chaotropic reagent (2M or 8M urea) thefluorescence intensity increase with time to a plateau as the moleculedenatured (unfolded).

These data indicate that the fluorogenic protease indicator normallyexists in a stable folded conformation created by the conformationdetermining regions, as was predicted by a model based on an energyminimization algorithm. The plateau fluorescence level representsresidual quenching of the fluorophores still joined by the fullydenatured peptide backbone. Digestion of the extended (denatured)peptide results in greater than a 2 fold increase in fluorescence as thefluorophores are able to move farther away from each other.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 56                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ProMetSerIle                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is norleucine."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProXaaSerIle                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlyArgThrGly                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgMetSerLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is norleucine."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgXaaSerLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlyArgSerLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuAlaLeuLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuGlyIleAla                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlnGlyIleLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlnGlyLeuLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlnGlyIleAla                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlnAlaIleAla                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GluGlyLeuArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyHisPheArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      LeuGluValMet                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(4)                                                       (D) OTHER INFORMATION: /note= "Xaa is norleucine."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      LeuGluValXaa                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      IleHisIleGln                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AlaAsnTyrAsn                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AlaGlyGluArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AlaGlyPheAla                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GlnGlyLeuAla                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AlaGlnPheVal                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AlaGlyValGlu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      PheCysMetLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is norleucine."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      PheCysXaaLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AspGlySerGlyGlyGlyGluAspGluLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      LysGluAspGlyGlyAspLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AspGlySerGlyGluAspGluLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      AspGlyGlyGlyLysLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      LysGluAspGluGlySerGlyAspLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(5)                                                       (D) OTHER INFORMATION: /note= "Xaa is norleucine."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      AspAlaIleProXaaSerIleProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      IleProMetSerIle                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AspAlaIleProMetSerIleProCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      LysLysGlyGlyGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(5)                                                       (D) OTHER INFORMATION: /note= "Xaa is Met or norleucine."                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AspAlaIleProXaaSerIleXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Met, aminoisobutryic aci                or Pro."                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Thr, aminoisobtyric acid                or Pro."                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AspXaaXaaGlyArgThrGlyXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Ala or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Thr, aminoisobutyric aci                or Pro."                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(6)                                                       (D) OTHER INFORMATION: /note= "Xaa is Met or norleucine."                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      AspXaaXaaPheCysXaaLeuXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is aminoisobutyric                         acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Ser, aminoisobutyric aci                or Pro."                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(5)                                                       (D) OTHER INFORMATION: /note= "Xaa is Met or norleucine."                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AspXaaXaaArgXaaSerLeuXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is aminoisobutyric                         acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Ala, aminoisobutyric aci                or Pro."                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      AspXaaXaaGlyArgSerLeuXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is aminoisobutyric                         acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Thr, aminoisobutyric aci                or Pro."                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AspXaaXaaLeuLeuSerLeuXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AspXaaXaaLeuGlyIleAlaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AspXaaXaaGlnGlyIleLeuXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      AspXaaXaaGlnGlyLeuLeuXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AspXaaXaaGlnGlyIleAlaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AspXaaXaaGlnAlaIleAlaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AspXaaXaaGluGlyLeuArgXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Asp, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Val, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AspXaaXaaGlyHisPheArgXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Asp, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Thr, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(7)                                                       (D) OTHER INFORMATION: /note= "Xaa is Met or norleucine."                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AspXaaXaaLeuGluValXaaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Arg, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Ala, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      AspXaaXaaIleHisIleGlnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      AspXaaXaaAlaGlyGluArgXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      AspXaaXaaAlaGlyPheAlaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      AspXaaXaaGlnGlyLeuAlaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Asp, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Val, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      AspXaaXaaAlaGlnPheValXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Asp or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      AspXaaXaaAlaGlyValGlnXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Gly, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      AspXaaXaaGlnGlyIleAlaXaaCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(2)                                                       (D) OTHER INFORMATION: /note= "Xaa is Asp, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(3)                                                       (D) OTHER INFORMATION: /note= "Xaa is Val, aminoisobutyric                    acid or Pro."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: one-of(8)                                                       (D) OTHER INFORMATION: /note= "Xaa is Pro or aminoisobutyric                  acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      AspXaaXaaAlaAsnTyrAsnXaaCys                                                   15                                                                            __________________________________________________________________________

What is claimed is:
 1. A fluorogenic composition for the detection ofthe activity of a protease, said composition having the formula:##STR13## wherein: Y is a composition selected from the group consistingof compounds of formulas: ##STR14## C¹ ₅, C¹ ₄, C¹ ₃, P₂, P₁, P'₁, P'₂,C² ₃, and C² ₄ are amino acids;P₂ -P₁ -P₁ '-P₂ ' comprises a proteasebinding site; F¹ and F² are fluorophores; S¹ and S² are peptide spacersranging in length from 1 to about 50 amino acids; n and k areindependently 0 or 1 and when n is 1, S¹ is joined to C¹ ₅ by a peptidebond through a terminal alpha amino group of C¹, and when k is 1, S² isjoined to C² ³ or to C² ₄ by a peptide bond through a terminal alphacarboxyl group of the respective C² ₃ or C² ₄ ; and wherein C¹ ₅ -C¹ ₄-C¹ ₃ and C² ₃ -C² ₄ or C² ₃ introduce a bend into the compositionthereby creating a generally U-shaped configuration of said compositionin which said fluorophores are adjacent to each other with a separationof less than about 100 Å.
 2. The composition of claim 1, in whichwhen P₂' is not a proline then Y is formula III in which C² ₃ -C² ₄ is Pro-Cysor α-aminoisobutyric acid (Aib)-Cys; when P₂ ' is a proline Y is formulaIV in which C² ₃ is Cys; when P₂ is a proline then C¹ ₅ -C¹ ₄ -C¹ ₃ isAsp-C¹ ₄ -C¹ ₃, or Asp-C¹ ₄ -Aib; and when P₂ is not a proline then C¹ ₅-C¹ ₄ -C¹ ₃ is selected from the group consisting of Asp-C¹ ₄ -Pro,Asp-C¹ ₄ -Aib, Asp-Aib-Pro, Asp-Pro-C¹ ₃, Asp-Pro-Aib and Asp-Aib-Aib.3. The composition of claim 2, whereinP₂ is selected from the groupconsisting of Pro, Gly, Phe, Arg, Leu, GIn, Glu, lie, and Ala; P₁ isselected from the group consisting of Cys, Met, norleucine (Nle), Gln,Arg, Leu, Gly, His, Glu, Ala, and Asn; P₁ ' is selected from the groupconsisting of Thr, Ser, Met, Nle, Leu, Ala, Ile, Phe, Vat, Glu, and Tyr;and P₂ ' is selected from the group consisting of Ile, Gly, Met, Nle,Leu, Ala, GIn, Arg, Val and Asn.
 4. The composition of claim 2,whereinC¹ ₅ is Asp; C¹ ₄ is selected from the group consisting of Ala,Met, norleucine (Nle), α-aminoisobutyric acid (Aib), Pro, Ile, Gly, Aspand Arg; and C¹ ₃ is selected from the group consisting of Ile, Aib andPro.
 5. The composition of claim 2, whereinP₂ ' is Pro; Y is Formula IV;C² ₃ is Cys.
 6. The composition of claim 2, whereinY is Formula III; C²₃ is Pro; and C² ₄ is Cys.
 7. The composition of claim 1, wherein F¹ isa fluorophore having an excitation wavelength ranging from 315 nm toabout 650 nm.
 8. The composition of claim 1, wherein F² is a fluorophorehaving an excitation wavelength ranging from 3 15 nm to about 650 nm. 9.The composition of claim 1, wherein F¹ is selected from the groupconsisting of 5-carboxytetramethylrhodamine and7-hydroxy-4-methylcoumarin-3-acetic acid.
 10. The composition of claim1, wherein F² is selected from the group consisting of rhodamine Xacetamide and7-diethylamino-3-((4'-iodoacetyl)amino)phenyl)-4-methylcoumarin.
 11. Thecomposition of claim 1, whereinn and k are 0; C¹ ₅ -C¹ ₄ -C¹ ₃ isAsp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ ' is Pro-norleucine (Nle)-Ser-Ile; C² ₃ -C²₄ is Pro-Cys; F¹ is 5-carboxytetramethylrhodamine; and F² is rhodamine Xacetamide.
 12. The composition of claim 1, whereinn and k are 0; C¹ ₅-C¹ ₄ -C¹ ₃ is Asp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ ' is Pro-Met-Ser-Ile; C² ₃-C² ₄ is Pro-Cys; F¹ is 5-carboxytetramethylrhodamine; and F² isrhodamine X acetamide.
 13. The composition of claim 1, whereinn is 1; kis 0; S¹ is Lys-Lys-Gly-Gly-Gly; C¹ ₅ -C¹ ₄ -C¹ ₃ s Asp-Ala-Ile; P₂ -P₁-P₁ '-P₂ ' is Pro-norleucine (Nle)-Ser-Ile; and C² ₃ -C² ₄ is Pro-Cys.14. The composition of claim 13, wherein S¹ is conjugated to a solidsupport.
 15. The composition of claim 1, whereinC¹ ₅ -C¹ ₄ -C¹ ₃ isAsp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ ' is Pro-norleucine (Nle)-Ser-Ile; and C² ₃-C² ₄ is Pro-Cys. n is 0; k is 1; and S² is Asp-Gly-Gly-Gly-Lys-Lys. 16.The composition of claim 15, wherein S² is conjugated to a solidsupport.
 17. A method of detecting the activity of a protease in abiological sample, said method comprising the steps of:contacting saidbiological sample with a fluorogenic composition having the formula:##STR15## wherein: Y is a composition selected from the group consistingof compounds of formulas: ##STR16## C¹ ₅, C¹ ₄, C¹ ₃, P₂, P₁, P'₁, P'₂,C² ₃, and C² ₄ are amino acids; P₂ -P₁ -P₁ '-P₂ ' comprises a proteasebinding site; F¹ and F² are fluorophores; S¹ and S² are peptide spacersranging in length from 1 to about 50 amino acids; n and k areindependently 0 or 1 and when n is 1, S¹ is joined to C¹ ₅ by a peptidebond through a terminal alpha amino group of C¹, and when k is 1, S² isjoined to C² ₃ or to C² ₄ by a peptide bond through a terminal alphacarboxyl group of the respective C² ₃ or C² ₄ ; and wherein C¹ ₅ -C¹ ₄-C¹ ₃ and C² ₃ -C² ₄ or C² ₃ introduce a bend into the compositionthereby creating a generally U-shaped configuration of said compositionin which said fluorophores are adjacent to each other with a separationof less than about 100 Å; and detecting a change in fluorescence of saidfluorogenic composition wherein an increase in fluorescence indicatesprotease activity.
 18. The method of claim 17, wherein said biologicalsample is a histological section that has not been fixed or imbedded.19. The method of claim 17, in whichwhen P₂ ' is not a proline then Y isformula III in which C² ₃ -C² ₄ is Pro-Cys or α-aminoisobutyric acid(Aib)-Cys; when P₂ ' is a proline Y is formula IV in which C² ₃ is Cys;when P₂ is a proline then C¹ ₅ -C¹ ₄ -C¹ ₃ is Asp-C¹ ₄ -C¹ ₃, or Asp-C¹₄ -Aib; and when P₂ is not a proline then C¹ ₅ -C¹ ₄ -C¹ ₃ is selectedfrom the group consisting of Asp-C¹ ₄ -Pro, Asp-C¹ ₄ -Aib, Asp-Aib-Pro,Asp-Pro-C¹ ₃, Asp-Pro-Aib and Asp-Aib-Aib.
 20. The method of claim 19,whereinP₂ is selected from the group consisting of Pro, Gly, Phe, Arg,Leu, GIn, Glu, Ile, and Ala; P₁ is selected from the group consisting ofCys, Met, norleucine (Nle), Gln, Arg, Leu, Gly, His, Glu, Ala, and Asn;P₁ ' is selected from the group consisting of Thr, Ser, Met, Nle, Leu,Ala, Ile, Phe, Val, Glu, and Tyr; and P₂ ' is selected from the groupconsisting of Ile, Gly, Met, Nle, Leu, Ala, Gln, Arg, Val and Asn. 21.The method of claim 20, whereinC¹ ₅ is Asp; C¹ ₄ is selected from thegroup consisting of Ala, Met, norleucine (Nle), α-aminoisobutyric acid(Aib), Pro, Ile, Gly, Asp and Arg; and C¹ ₃ is selected from the groupconsisting of Ile, Aib and Pro.
 22. The method of claim 20, whereinP₂ 'is Pro; Y is Formula IV; C² ₃ is Cys.
 23. The method of claim 20,whereinY is Formula III; C² ₃ is Pro; and C² ₄ is Cys.
 24. The method ofclaim 17, wherein F¹ is a fluorophore having an excitation wavelengthranging from about 3 15 nm to about 650 nm.
 25. The method of claim 17,wherein F² is a fluorophore having an excitation wavelength ranging fromabout 3 15 nm to about 650 nm.
 26. The method of claim 17, wherein F¹ isselected from the group consisting of 5-carboxytetramethylrhodamine and7-hydroxy-4-methylcoumarin-3-acetic acid.
 27. The method of claim 17,wherein F² is selected from the group consisting of rhodamine Xacetamide and7-diethylamino-3-((4'-iodoacetyl)amino)phenyl)-4-methylcoumarin.
 28. Themethod of claim 17, whereinn and k are 0; C¹ ₅ -C¹ ₄ -C¹ ₃ isAsp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ ' is Pro-Nle-Ser-Ile; C² ₃ -C² ₄ isPro-Cys; F¹ is 5-carboxytetramethylrhodamine; and F² is rhodamine Xacetamide.
 29. The method of claim 17, whereinn and k are 0; C¹ ₅ -C¹ ₄-C¹ ₃ is Asp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ ' is Pro-Met-Ser-Ile; C² ₃ -C² ₄is Pro-Cys; F¹ is 5-carboxytetramethylrhodamine; and F² is rhodamine Xacetamide.
 30. The method of claim 17, whereinn is 1; k is 0; S¹ isLys-Lys-Gly-Gly-Gly; C¹ ₅ -C¹ ₄ -C¹ ₃ is Asp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ 'is Pro-Nle-Ser-Ile; and C² ₃ -C² ₄ is Pro-Cys.
 31. The method of claim30, wherein S¹ is conjugated to a solid support.
 32. The method of claim17, whereinC¹ ₅ -C¹ ₄ -C¹ ₃ is Asp-Ala-Ile; P₂ -P₁ -P₁ '-P₂ ' isPro-Nle-Ser-Ile; and C² ₃ -C² ₄ is Pro-Cys. n is 0; k is 1; and S² isAsp-Gly-Gly-Gly-Lys-Lys.
 33. The method of claim 32, wherein S² isconjugated to a solid support.